Metformin attenuates osteoclast-mediated abnormal subchondral bone remodeling and alleviates osteoarthritis via AMPK/NF-κB/ERK signaling pathway

This study explored the mechanism by which metformin (Met) inhibits osteoclast activation and determined its effects on osteoarthritis (OA) mice. Bone marrow-derived macrophages were isolated. Osteoclastogenesis was detected using tartrate-resistant acid phosphatase (TRAP) staining. Cell proliferation was evaluated using CCK-8, F-actin rings were detected by immunofluorescence staining, and bone resorption was detected using bone slices. Nuclear factor kappa-B (NF-κB) and nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) were detected using luciferase assays, and the adenosine monophosphate-activated protein kinase (AMPK), NF-κB, and mitogen-activated protein kinase (MAPK) signaling pathways were detected using western blotting. Finally, expression of genes involved in osteoclastogenesis was measured using quantitative polymerase chain reaction. A knee OA mouse model was established by destabilization of the medial meniscus (DMM). Male C57BL/6J mice were assigned to sham-operated, DMM+vehicle, and DMM+Met groups. Met (100 mg/kg/d) or vehicle was administered from the first day postoperative until sacrifice. At 4- and 8-week post OA induction, micro-computed tomography was performed to analyze microstructural changes in the subchondral bone, hematoxylin and eosin staining and Safranin-O/Fast Green staining were performed to evaluate the degenerated cartilage, TRAP-stained osteoclasts were enumerated, and receptor activator of nuclear factor κB ligand (RANKL), AMPK, and NF-κB were detected using immunohistochemistry. BMM proliferation was not affected by Met treatment below 2 mM. Met inhibited osteoclast formation and bone resorption in a dose-dependent manner in vitro. Met suppressed RANKL-induced activation of p-AMPK, NF-κB, phosphorylated extracellular regulated protein kinases (p-ERK) and up-regulation of genes involved in osteoclastogenesis. Met reversed decreases in BV/TV, Tb.Th, Tb.N, and CD, and an increase in Tb.Sp at 4 weeks postoperatively. The number of osteoclasts and OARSI score were decreased by Met without effect on body weight or blood glucose levels. Met inhibited RANKL, p-AMPK, and NF-κB expression in early OA. The mechanism by which Met inhibits osteoclast activation may be associated with AMPK/NF-κB/ERK signaling pathway, indicating a novel strategy for OA treatment.

Introduction Osteoarthritis (OA) is the most common degenerative disease involving multiple joints and seriously affects the quality of life of patients. The main causes of OA include age, obesity, mechanical factors, and genetic predisposition [1]. With global increases in life expectancies, the prevalence of OA is increasing every year [2]. Currently, the pathogenesis of OA is not completely understood, which hinders a complete cure from being achieved. Patients with advanced stage OA often cannot avoid the need of surgical treatment, which is responsible for serious physical and economic burden to both the individual patient and society in general [3]. Therefore, OA and its treatment have become an urgent social problem. It is of great relevance to investigate the pathogenesis of OA in order to identify precise and targeted treatments.
As a structural and functional unit, articular cartilage and subchondral bone play an important role in the development and progression of OA [4]. Multiple studies have demonstrated that abnormal subchondral bone remodeling occurs prior to the degeneration of articular cartilage during OA [5,6]. Under normal physiological conditions, a balance in subchondral bone remodeling is maintained through osteoblast-mediated bone formation and osteoclastmediated bone resorption [7]. However, during the early stage of OA, when the joint is subjected to abnormal mechanical stimulation, osteoclast-mediated bone absorption is enhanced. This leads to reduced bone mass and destruction of the microstructure in the subchondral bone, thereby increasing the risk of degeneration of the overburdened articular cartilage. With changes in the mechanical and biochemical microenvironment of the subchondral bone, osteosclerosis occurs and the microstructure is further damaged, aggravating cartilage degeneration [8].
The biguanide metformin (Met) is the first-line drug of choice for the treatment of type 2 diabetes [14]. In addition, multiple studies have demonstrated that Met has beneficial effects on several age-related diseases [15,16]. Met can affect a variety of metabolic and cellular physiological processes, including proliferation, inflammation, oxidative damage, autophagy, cell apoptosis, and senescence [17][18][19]. Fan et al. showed in a collagen-induced arthritis (CIA) rat model that Met can suppress systemic inflammation and synovitis, and protect bone by inhibiting osteoclast formation, extracellular matrix (ECM) degradation, and chondrocyte apoptosis [20]. Met can also reduce the risk of osteoporosis and osteoporotic fractures in patients with diabetes through a direct osteogenic effect on osteoblasts [21].
As the main sensor of cellular energy status [22], it is widely believed that adenosine monophosphate-activated protein kinase (AMPK) plays a significant role in regulating the microenvironment and biological behavior of cells. Yan et al. showed that Met can activate AMPK and attenuate implant debris-induced osteolysis by reducing osteoclast numbers and the polarization of macrophages to an anti-inflammatory functional phenotype [23]. However, the effect and mechanism of Met on abnormal subchondral bone remodeling induced by osteoclasts during the early stages of OA remain unclear. Therefore, based on previous studies, we speculate that Met may be an activator of AMPK. In the present study, we investigated the effect and possible mechanism of Met on osteoclast formation and bone resorption in vitro. We also examined the therapeutic effect of Met using a destabilization of the medial meniscus (DMM)induced OA mouse model to further elucidate whether Met may be a candidate therapeutic agent for treating OA.

Osteoclastogenesis assay in vitro
BMMs were seeded into a 96-well plate at a density of 5 × 10 3 /well and incubated 7 d with α-MEM containing 30 ng/mL M-CSF, 50 ng/mL RANKL (390-TN-010; R&D Systems), and various concentrations of Met (0, 0.1, 0.5, 1, and 2 mM). After washing with phosphate-buffered saline (PBS) and fixing with 4% paraformaldehyde for 20 min, the cells were stained with TRAP solution (PMC-AK04F-COS; Cosmo Bio Co., Tokyo, Japan) for 5 min. TRAP-positive cells with more than three nuclei were identified as mature osteoclasts, and the number and area of osteoclasts in five fields of view per well were measured using an Olympus DP71 light microscope [25].

Bone resorption assay
BMMs were seeded into 96-well plates at a density of 5 × 10 3 /well along with bone slices (DT-1BON1000; Immunodiagnostic Systems, Herlev Hovedgade, Denmark) and cultured for 9 d in medium containing 30 ng/mL M-CSF, 50 ng/mL RANKL, and different concentrations of Met (0, 0.5, and 2 mM). After washing with PBS and fixing with 2.5% glutaraldehyde at 4˚C for 7 min, the bone slices were placed in 0.25 M ammonium hydroxide, sonicated to remove the cells, washed in distilled water, and then dehydrated using graded alcohols for 10 min. After air-drying, the bone slices were stained with 0.5% (w/v) aqueous toluidine blue (G3668; Solarbio Co.) for 3 min, and the bone resorption areas were examined by light microscopy. To detect resorption pits, bone slices, after washing and dehydration as described above, were fixed in 2.5% glutaraldehyde and 1% osmic acid for 2 h, dried using graded tert-butyl alcohol, and then sputtered with gold using an airless spray unit. A Hitachi S-3400N scanning electron microscope (SEM; Hitachi, Tokyo, Japan) was used to observe and count the number of resorption pits in five fields of view on the bone slices [26].

Luciferase assay
Murine macrophage RAW264.7 cells (American Type Culture Collection, Rockville, MD, USA) were stably transfected by electroporation with 2 μg pcDNA3.1 and 20 μg 3kB-Luc-SV40 reporter construct. To investigate NF-κB and NFATc1 activation, cells were transfected with NF-κB, NFATc1, and β-galactosidase and selected using 400 μg/ml of G418 (10131035; Thermo Fisher Scientific) as described previously [25,27]. Briefly, the transfected RAW264.7 cells were seeded into 24-well plates at a density of 5 × 10 5 or 1 × 10 5 cells/well in triplicate and cultured. After 24 h, the cells were pretreated with 0, 0.1, 0.5, 1, or 2 mM Met for 2 h, and then incubated for 6 h with α-MEM containing 50 ng/mL RANKL to activate NF-κB or for 24 h to activate NFATc1. After activation, the cells were washed with PBS, lysed with Cell Culture Lysis Reagent (120 μL/well) at 4˚C for 10 min, and the supernatant collected after centrifugation at 10,000 × g for 20 min. A Luciferase Assay Kit (E1483; Promega, Madison, WI, USA) was used to detect NF-κB and NFATc1 activity. The data were normalized to β-galactosidase activity for each sample.

RNA extraction and reverse transcription quantitative PCR (RT-qPCR) assay
BMMs were seeded into 6-well plates at a density of 1 × 10 6 cells/well and cultured for 24 h to reach confluence. The cells were then incubated for 7 d in α-MEM containing 30 ng/mL M-CSF, 50 ng/mL RANKL, and 0, 0.1, 0.5, 1, or 2 mM Met. Multisource Total RNA Prep Kits (AP-MN-MS-RNA-250, Axygen, Union City, CA, USA) were used to extract total RNA. Complementary DNA (cDNA) was synthesized from 1 μg total RNA using a TransScript 1 All-in-One First-Strand cDNA Synthesis Kit (AT341-01; TransGen Biotech) as described by the manufacturer's instructions. RT-qPCR was subsequently performed to quantitate the expression of genes related to osteoclastogenesis. The RT-qPCR cycling conditions included 45 cycles of 94˚C for 30 s, 54˚C for 30 s, and 72˚C for 34 s. The sequences of the primers are listed in Table 1. All reactions were run in triplicate and β-actin was used as the quantitative internal control gene.

Ethics approval
C57BL/6 mice (protocol no. 2020-0001) were provided by the Laboratory Animal Center of Ningxia Medical University. The operation of animal experiments (approval no. LACUC-N-LAC-2020-115) conformed to the Laboratory Animal Ethical and Welfare Committee of Laboratory Animal Center, Ningxia Medical University. All animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All surgeries were performed under anesthesia, all mice were euthanized via an overdose of intraperitoneal sodium pentobarbital, and the three "Rs" principle were strictly followed to minimize number and suffering of mice in this experiment, including i) Reduce-use fewer animals, ii) Refine-enhance efficiency of the experimental protocol, iii) Replace-employ in vitro over in vivo whenever possible.

Animals
A total of 30 male 10-week-old C57BL/6J mice weighing 24.7 ± 1.9 g were provided by the Laboratory Animal Center of Ningxia Medical University, Yinchuan, China (protocol no. 2020-0001). The mice were group-housed under standard laboratory conditions with a 12-h light/ dark cycle, a constant temperature of 25˚C and humidity of 48%, and standard food and water ad libitum. The mice were randomly assigned to three groups (n = 10 mice/group), the sham-operated group, DMM+vehicle group, and DMM+Met group. The mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium in PBS (60 mg/kg) and received surgical treatment as previously described to establish the OA model [28]. Briefly, the right knee joint capsule was exposed using a medial parapatellar approach. The medial meniscotibial ligament was then transected using micro-scissors and the medial meniscus reflected proximally toward the femur. The joint capsule and skin were sutured with 5-0 synthetic absorbable suture. Mice in the sham-operated group were similarly treated but without transection of the medial meniscotibial ligament or medial meniscus. The DMM+Met group received a supplemental treatment of 100 mg/kg/d Met via orogastric intubation starting from the first day after surgery until sacrifice (4-wk and 8-wk post operative). The sham-operated and DMM+vehicle groups received orogastric intubation with an equal volume of distilled water as a control. The experimental design of the animal studies is shown in Fig 1.

Body weight and blood glucose level measurement
To determine whether the application of Met affects body weight and blood glucose levels in mice, we measured the body weight and random blood glucose levels of the animals once a week, as previously described [29]. After blood was collected from the caudal vein, an

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ACCU-CHEK Active Glucometer (Roche Diagnostic, Mannheim, Germany) was used to evaluate the blood glucose levels of each group.

Micro-computed tomography (CT) analysis
A Skyscan 1176 μCT (Bruker, Billerica, MA, USA) was used to evaluate the microarchitecture of the tibial subchondral bone, as described previously [30]. Briefly, the knee joints of five animals from each group were collected and excess soft tissue removed. After fixation with 4% paraformaldehyde for 36 h at 20˚C, the proximal tibiae were imaged by μCT under the following parameter settings: resolution, 5 μm/pixel; 50 kV and 250 μA; exposure time, 1.6 sec; 0.6å ngular step; and 180˚scan. A portion of the load-bearing region at the medial tibial plateau 1 mm ventrodorsal in length, 0.2 mm below the growth plate, and 0.5 mm in height was identified as the region of interest (ROI) for analysis of bone parameters. The parameters considered included bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), and connectivity density (CD).

Histology and immunohistochemistry assay
After μCT analysis, the five knee joints from each group were decalcified with 10% disodium ethylenediaminetetraacetate dihydrate (E8030; Solarbio Co.) for 3 wk at 20˚C. The decalcified specimens were dehydrated, cleared, embedded in paraffin, and sectioned at 5 μm thickness along the sagittal plane. The sections were stained using a hematoxylin and eosin (HE) staining kit (G1005; Servicebio, Wuhan, China) and examined to determine the thickness of calcified cartilage (CC) and hyaline cartilage layers, as described previously [31]. Briefly, the slices were dewaxed in xylene twice for 10 min, followed by treatment in anhydrous ethanol twice for 10 min each, and 75% alcohol for 7 min each. The sections were stained with HE for 5 min and then dehydrated in 85% and 95% alcohol for 10 min each. For staining with Safranin-O/Fast Green and TRAP (G1053 and G1050, respectively; Servicebio), tissue sections on the slides were deparaffinized as described above and subsequently stained with Fast Green for 5 min, washed, dehydrated, and counter-stained with Safranin-O for 3 min. The degeneration grade of articular cartilage was evaluated according to the Osteoarthritis Research Society International (OARSI) scoring system, as described previously [32]. For TRAP staining, the slides were washed three times after incubation for 2 h with the working solution provided with the kit and then stained with hematoxylin for 5 min. The ratios of Oc.S/BS in five fields of view were detected using the Olympus DP71 light microscope [33]. For immunohistochemical staining, slides from each group 4-wk postoperative were incubated with 3% hydrogen peroxide for 15 min and then blocked with 5% goat serum for 1 h at 37˚C. The slides were then incubated for 10 h at 4˚C with primary antibodies against RANKL (1:100, 55184-1-AP; Proteintech Group), NF-κB (1:100, 66553-1-Ig; Proteintech Group), and p-AMPK (1:200, ab133448; Abcam). After washing with PBS, the slides were incubated with a secondary antibody for 1 h at 20˚C and color was developed using 3,3-diaminobenzidine (DAB) (ZLI-9018; Zhongshan Jinqiao Biotechnology Co.) as a chromogen prior to counterstaining with hematoxylin. The Olympus DP71 microscope was used to observe the positivestaining cells in five fields of view.

Statistical analysis
GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA) was used for the statistical analyses. Experiments were repeated at least three times and the data were expressed as the mean ± standard deviation. One-way analysis of variance (ANOVA) followed by the Tukey post-hoc test was performed to compare data among three or more groups. A student's t-test was used to compare data between two groups. P < 0.05 was considered to indicate a statistically significant difference.

Isolation and culture of primary BMMs, and osteoclast differentiation and identification
After overnight incubation, BMMs were induced for osteoclast differentiation in α-MEM containing M-CSF (30 ng/ml) and RANKL (50 ng/ml). After induction for 3 d, BMMs were mostly mononuclear and oval in shape (Fig 2A). The cells then gradually demonstrated a colony distribution (Fig 2B). After induction for 7 d, the cells fused with each other and had multiple nuclei. The multinucleated giant cells were surrounded by F-actin rings, which could be clearly displayed by TRAP staining (Fig 2C) and TRITC-conjugated phalloidin staining ( Fig  2D). These results indicated that BMMs could be successfully obtained using our methodology and could be induced to differentiate into osteoclasts.

Effect of Met on BMMs proliferation
After overnight incubation, BMMs were cultured in α-MEM containing various concentrations of Met (0, 0.1, 0.5, 1, 2, and 5 mM) for 72 h. Absorbance values detected from CCK-8 assays revealed that BMM proliferation was not affected by Met treatment when the concentration was below 2 mM; however, BMM proliferation was significantly inhibited by Met once the concentration reached 5 mM (Fig 2E). Accordingly, Met concentrations of 0-2 mM were selected to investigate the effect of Met on osteoclast differentiation and bone resorption.

Met inhibited the differentiation of BMMs into osteoclasts in vitro
To investigate the effect of Met on osteoclast formation, BMMs were cultured for 7 d in α-MEM containing M-CSF and RANKL in the absence or presence of Met at various concentrations. As shown by TRAP staining, Met significantly suppressed osteoclast formation in a dose-dependent manner (Fig 3A). The number of osteoclasts, defined by being TRAP-positive with more than three nuclei (black arrows), was 185.5 ± 4.36 per well in the group stimulated without Met and 19.49 ± 2.19 per well in the group stimulated with 2 mM Met (Fig 3B). Osteoclast formation based on TRAP staining was evaluated at different time points and was found to be significantly inhibited after 3 d of stimulation with 2 mM Met (Fig 3C). The numbers and areas of osteoclasts further demonstrated that Met suppressed osteoclastogenesis at an early stage (Fig 3D).
To further investigate the effects of Met on osteoclastogenesis and osteoclast function, RT-qPCR was performed to determine the expression level of osteoclast formation-related and resorption-related genes. The results indicated that the expression of genes such as RANK, TRAP, CTSK, CTR, MMP-9, and DC-STAMP were all upregulated in response to RANKL stimulation. However, a dose-dependent inhibition of gene expression was observed in the groups treated with the different concentrations of Met (Fig 3E).

Met restrained F-actin rings formation and bone resorption of osteoclast in vitro
The structures of F-actin rings in osteoclasts induced by RANKL were visualized by immunofluorescence staining using TRITC-conjugated phalloidin. However, the number and morphology of F-actin rings in Met-treated osteoclasts were significantly altered (Fig 4A). For instance, the mean numbers of F-actin rings per microscopic field of view were 12.64 ± 1.152 in the group without Met, 7.05 ± 0.74 in the group treated with 0.5 mM Met, and 3.18 ± 0.85 in the group treated with 2 mM Met (Fig 4D). These results indicate that Met restrained Factin ring formation in osteoclasts in vitro.
Toluidine blue staining and SEM analysis revealed large areas and numbers of bone resorption pits on bone slices stimulated with M-CSF and RANKL but not treated with Met compared with that of groups of bone slices stimulated and also treated with Met, which exhibited smaller areas and fewer numbers of bone resorption pits (Fig 4B and 4C). The percentages of resorption area decreased from 31.39 ± 2.60 in the group not treated with Met to 21.95 ± 2.18 and < 9.40 ± 1.50 after treatment with 0.5 mM and 2 mM Met, respectively (Fig 4E). The number of resorption pits/mm 2 was reduced from 57.77 ± 2.96 in the group not treated with Met to 12.76 ± 2.46 after treatment with 2 mM Met (Fig 4F). These results suggest that Met inhibited osteoclast bone resorption in vitro.

Met inhibited RANKL-induced AMPK, NF-κB and ERK activation during osteoclastogenesis
As demonstrated by western blotting, decreases in p-AMPK and IκBα levels were accompanied by increases in NF-κB, p-ERK1/2, p-JNK, and p-p38 levels during RANKL-induced osteoclastogenesis, while pretreatment with Met reversed this trend. The decrease of p-AMPK levels was blocked by 2 mM Met (Fig 5A and 5B). Meanwhile, IκBα degradation and NF-κB activation were significantly inhibited at 30 min poststimulation of RANKL (Fig 5C and 5D). To further investigate whether Met affected transcription factors related to osteoclast differentiation, RAW264.7 cells were transfected with luciferase reporter constructs in order to evaluate the activities of NF-κB and NFATc1. The findings showed that 0.1 to 2 mM Met

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significantly inhibited luciferase activities of NF-κB and NFATc1 in a dose-dependent manner (Fig 5E and 5F). Pretreatment with 2 mM Met significantly inhibited the expression of p-ERK1/2 at 30 min after RANKL stimulation, but had no influence on p-JNK or p-p38 expression (Fig 5G and 5H). Together, these results demonstrate that Met suppressed RANKLinduced osteoclastogenesis via the AMPK/NF-κB/ERK signaling pathway.

Met attenuated bone loss during the early stage of DMM-induced OA
The DMM-induced OA mice were treated with Met (100 mg/kg/d) or the vehicle alone by intragastric administration for 4 wk or 8 wk and μCT was then performed to examine the microstructure of the tibial subchondral bone (Fig 6A). The results based on the microstructural parameters evaluated indicated there was significant bone loss in the DMM+vehicle group compared with that in the sham-operated group. Specifically, there were decreases in BV/TV (Fig 6B), Tb.Th (Fig 6C), Tb.N (Fig 6D), and CD (Fig 6F), and an increase in Tb.Sp (Fig 6E) at 4 weeks postoperatively. However, these changes were reversed by intragastric administration of 100 mg/kg/d Met for 4 weeks and 8 weeks; Met had no effect on body weight (Fig 6G) or blood glucose levels ( Fig 6H) in either group. These results indicate that Met was able to alleviate bone loss without affecting body weight or blood glucose levels during the early stage of OA.

Met suppressed osteoclastogenesis in the early stage of DMM-induced OA
As revealed by TRAP staining of tibial subchondral bone, the ratio of Oc.S/BS was 31.51 ± 3.241 in the DMM+vehicle group at 4 weeks postoperatively, which was higher than that in the sham-operated group, whereas Met significantly decreased the ratio to 12.41 ± 2.62 (Fig 7A and 7D). These results confirmed the subchondral bone resorption detected by μCT and support the concept that DMM enhanced subchondral bone resorption by promoting osteoclast formation (black arrows) during the early phase of OA.

Met inhibited articular cartilage degeneration in DMM-induced OA mouse
Both HE and Safranin-O/Fast Green staining demonstrated that cartilage degeneration generally occurred after DMM surgery and gradually worsened in a time-dependent manner. HE staining revealed that the HC (white lines) thickness in the DMM+vehicle group decreased compared with that in the sham-operated group, while the CC (black lines) thickness increased and moved closer to the articular surface (Fig 7B). The CC/TAC ratio was 91.67 ± 4.51 in DMM+vehicle group at 8 weeks postoperatively, which was higher than that in sham-operated group, whereas Met significantly decreased the ratio to 64.77 ± 3.51 (Fig 7E).
Safranin-O/Fast Green staining showed that the matrix and chondrocytes in the DMM+vehicle group were degraded and irregular cracks were generated in cartilage at 4 weeks postoperatively compared to that of the sham-operated control group, while it became more widespread at 8 weeks postoperatively and full-thickness degeneration of the cartilage occurred with the development of OA (Fig 7C). However, Met significantly alleviated cartilage degeneration and delayed the process of OA. The OARSI score was 6.04 ± 0.57 in DMM +vehicle group at 8 weeks postoperatively, which was higher than that in sham-operated group, whereas Met significantly decreased the score to 2.98 ± 0.35 (Fig 7F).

Met inhibited RANKL, NF-κB expression and promoted p-AMPK expression in the early stage of DMM-induced OA
As shown by the immunohistochemical staining, there were more cells (black arrows) positive for RANKL (Fig 8A), NF-κB ( Fig 8E) and less cells positive for p-AMPK (Fig 8C) in the DMM +vehicle group compared with that in the sham-operated group. However, Met decreased the percentages of cells positive for RANKL (Fig 8B), NF-κB (Fig 8F) induced by DMM and increased the percentage of cells positive for p-AMPK (Fig 8D). The percentages of cells positive for RANKL, p-AMPK, and NF-κB were 90.21 ± 3.74, 21.23 ± 2.34, and 80.75 ± 4.21 respectively in DMM+vehicle group at 4 weeks postoperatively, whereas Met significantly revised them to 19.42 ± 3.14, 65.02 ± 3.65, and 8.74 ± 2.97. These results further confirmed the inhibitory effect of Met on osteoclast formation and function.

Discussion
OA is a chronic progressive degenerative disease that is closely associated with age. OA is the most common form of arthritis and a major contributor to chronic dysfunction in the elderly, affecting on both the physical and mental health of individuals. The prevalence of patients affected by symptomatic OA is increasing due to the aging of the population and the epidemic of obesity [2]. Subchondral bone remodeling plays an important role in the occurrence and development of OA. In early OA, osteoclast-mediated bone resorption increases and bone mass decreases, while in advanced OA, osteoblast-mediated bone formation increases and osteosclerosis occurs. The structural changes lead to the failure of cartilage support needed for adaptive support and shock absorption, leading to cartilage degeneration [34]. Based on the spatiotemporal progression of OA, Cai et al. attempted to apply drugs, such as bisphosphonates, at the early stage of OA in effort to inhibit osteoclast-mediated bone resorption, alleviate abnormal subchondral bone remodeling, delay the progression of OA, and achieve positive results, providing a new strategy for the early treatment of OA [35]. The increased prevalence of OA in patients with type 2 diabetes suggests that OA may be associated with energy metabolism [36]. Multiple studies have shown that Met, in addition to regulating blood glucose, is able to regulate bone remodeling and it exhibits osteogenic and chondroprotective effects [37][38][39]. However, little is known regarding the effects of Met on osteoclast formation and it potential as an OA treatment. Therefore, we designed the current study to investigate the effect and mechanism of Met on osteoclast formation and evaluated it in DMM-induced OA mice. We found that Met suppressed osteoclast formation and bone resorption both in vitro and in vivo by inhibiting AMPK/NF-κB/ERK signaling, and also alleviated osteoclast-mediated abnormal bone resorption, and delayed OA progression.
OA is a disease that affects all joints. Articular cartilage and subchondral bone form a structural and functional unit through crosstalk and jointly participate in the occurrence and development of OA [4]. It has been shown that osteoclast-mediated enhancement of bone resorption in subchondral bone in the early stage of OA changes the microstructure and microenvironment of the subchondral bone, resulting in the progressive degeneration of with NF-κB and NFATc1 were pretreated with various concentrations of Met for 2 h, and then incubated with α-MEM containing 30 ng/ml M-CSF and 50 ng/mL RANKL for 6 h to activate NF-κB and 24 h to activate NFATc1. Luciferase activities of (E) NF-κB and (F) NFATc1 were quantitatively analyzed. n = 3 per group. �� P < 0.01 and ��� P < 0.001, compared with control group.
https://doi.org/10.1371/journal.pone.0261127.g005 articular cartilage that is observed in advanced OA [40], which is consistent with our previous studies [41]. In the current study, we established an OA model using the DMM method. Compared to the anterior cruciate ligament transection (ACLT) method, the DMM method is minimally invasive and can avoid the interference of trauma and an acute inflammatory response and can better simulate the pathological changes of early OA [42]. The μCT results demonstrated there was abnormal subchondral bone remodeling as represented by a turnover from enhanced bone loss at 4 weeks postoperatively to enhanced osteosclerosis at 8 weeks postoperatively, which was consistent with the number of osteoclasts stained by TRAP in subchondral bone and the OARSI scores represented by Safranin O-Fast Green staining. Therefore, we considered 4 weeks postoperatively as a period of bone resorption. However, the application of Met inhibited bone loss and osteoclast formation in the subchondral bone, alleviating degeneration of the articular cartilage. This may have been due to the spatiotemporal effect of Met on OA, which delays the progression of OA by inhibiting the occurrence of bone resorption. Met may also have a direct protective effect on articular cartilage [43].

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As a master regulator of energy balance and metabolism, AMPK has been shown to be linked to multiple age-related diseases, including diabetes, cardiovascular disease, cancer, and OA [44]. Decreased phosphorylation of AMPK is observed in cartilage from both humans and mice with OA [45,46]. Wang et al. demonstrated that NF-κB signaling can be inhibited by upregulating AMPK expression to alleviate inflammation in the testes of aging rats [47]. Multiple studies have also demonstrated that Met plays a protective role as an AMPK activator in a number of diseases. For instance, Li et al. found that Met significantly inhibits the increased pain sensitivity caused by DMM surgery by upregulating AMPK expression [43].
In the early stage of OA, the bone marrow microenvironment in subchondral bone changes and BMMs differentiate into osteoclasts under the influence of biomolecules, including those of the RANKL/RANK/OPG axis, NF-κB, ERK, JNK, and p38 [48,49]. In the present study, we demonstrated that Met significantly suppressed osteoclast formation and bone resorption in a dose-dependent manner, accompanied by the inhibition of expression of osteoclast marker genes, such as RANK, TRAP, Ctsk, CTR, MMP-9, and DC-STAMP. Our western blotting results showed that in vitro Met upregulated the phosphorylation of AMPK and inhibited the expression of NF-κB during RANKL-induced osteoclast differentiation, which was consistent with the immunohistochemistry and luciferase assay results. However, the western blotting results demonstrated that Met only inhibited the phosphorylation of ERK and had no significant effect on JNK or p38 phosphorylation, which differed from the results of other studies. Yu et al. showed that RANKL-induced osteoclast differentiation was inhibited by suppression of ERK, JNK, and p38 phosphorylation in mouse BMMs [50,51]. This difference may be related to the fact that OA is a heterogeneous disease and the experimental progression of OA is affected by many factors, including gender, age, species, and the specific modeling methods employed [42,52]. Therefore, male C57BL/6J mice were selected to establish the OA model in our current study in order to avoid the influence of changes in estrogen levels. On the other hand, the observed differences may also indicate that different drugs have different targets for the same disease.

Conclusion
Our present study demonstrated that Met suppressed RANKL-induced differentiation of BMMs into osteoclasts and alleviated cartilage degeneration in DMM-induced OA mice. The inhibitory effect appeared to occur predominantly during the early stage of RANKL-induced osteoclast formation. Increases in p-AMPK levels and suppression of NF-κB and p-ERK levels accounted, at least in part, for the mechanism of action (Fig 9).